The Tox21 programs federal partners include the Environmental Protection Agency (EPA), the Food and Drug Administration (FDA) and NIH, with leadership from NCATS and the National Toxicology Program (NTP) at the National Institute of Environmental Health Sciences (NIEHS). These agencies work together to advance in vitro toxicological testing. The Tox21 Program is comprised of three NCATS teams: Systems Toxicology, Genomic Toxicology, and Computational Toxicology. The Systems Toxicology team has identified, developed, optimized, and/or screened more than 19 assays. Highlights range from performing 10 online screens, including estrogen-related receptor (ERR)/proliferator-activated-receptor-gamma coactivator (ERR/PGC), thyroid stimulating hormone receptor (TSHR) and histone deacetylase I/II (HDAC I/II) assays in triplicate against the Tox21 10K compound collection and 9 online validation assays, including cell-based acetylcholinesterase (AChE) assay, signaling pathway and nuclear receptor assays, against the LOPAC collection on the Tox21 robotic system. All of these assays were optimized and evaluated before moving to robotic online validation and online screening. AChE is the primary cholinesterase in the body that metabolizes a key neurotransmitter, acetylcholine. Inhibition of AChE activity can lead to neurotoxicity and known inhibitors include organophosphorus pesticides, chemical warfare agents, drugs, and various phytochemicals. In collaboration with the CFSAN/FDA, the Systems Toxicology team has developed a cell-based AChE assay in a homogenous format and validated this assay by screening the LOPAC library. The team has also identified a group of androgen-active and inactive compounds from the Tox21 10K screening. Several follow-up studies have been performed, including a biochemical assay determining the binding affinity, as well as a translocation assay to identify AR translocation into the nucleus. In addition, the team has worked on the mitochondria project and selected about 800 compounds to be tested in rat hepatocyte MMP, reactive oxygen species (ROS), and ATP content assays. About 60 compounds were further tested in pathway assays, C. elegans models, mitochondrial gene expression experiment using RASL-seq, and oxygen consumption assays. In collaboration with NIEHS, we optimized and screened PGC-ERR and ERR luciferase reporter gene assays against Tox21 10K compound collection. After the primary screening, 1000 compounds were cherry picked and re-tested in the ERR and PGC-ERR assays. The manuscript of assay development and validation has been submitted. The team has also searched for epigenetic assays for the Tox21 Program by optimizing a cell-based HDAC I/II assay in various cancer cell and stem cell backgrounds and validating this assay by screening the NCATS Pharmaceutical Collection of 2,816 approved and investigational drugs. The compounds were also tested with a control assay to identify false positives as results of luciferase inhibition or protease inhibition. Using an orthogonal HDAC biochemical assay, we profiled compound selectivity of 30 identified HDAC inhibitors against a panel of HDAC subtypes. The manuscript with these finding has been published in Journal of Biomolecular Screening this year. The TRHR project is a collaborative effort with NCATS and the EPA to generate and evaluate the TRHR cell line by measuring calcium levels after TRH stimulation. This assay has been validated offline and is ready for online validation. In addition, the team has identified a group of CAR activators and deactivators from the primary screening. Several follow-up studies have been performed to confirm the compounds with CAR activity. In order to continue the development biology project funded through NCS, Xias group has profiled the effect of the chemicals on the neural stem cells derived from normal, autistic, and schizophrenic patients in collaboration with the Johns Hopkins University.